PROJECT SUMMARY Immunotherapy treatments can be life saving for cancer patients, but only a small fraction of patients respond to this therapy. There have been no studies to date describing the characteristics of patients that respond compared to non-responders. Obesity and advanced age are important cancer risk factors, and both of these factors contribute to substantial immune dysfunction in patients. In obesity, there are more adipose tissue macrophages than in lean individuals, and these macrophages are of a pro-inflammatory ?M1? phenotype, whereas most lean individuals have pre-dominantly ?M2? anti-inflammatory macrophages. However, the change in macrophage polarization from an adipose tissue microenvironment to a tumor microenvironment is poorly described. Understanding if the tumor associated macrophages in obesity maintain their ?M1? phenotype or change to a tumor association macrophage ?M2? phenotype is critical in hypothesizing if obese patients will respond more or less effectively to immunotherapy. Additionally, many tumors in obese patients have higher mutational loads due to increased cellular reactive oxygen species, such as higher rates of genomic instability in colon cancer and endometrial cancer. Higher mutational loads are a prognostic factor positively correlated with immunotherapy efficacy, since the tumors are more immunogenic. Given these opposing obesity-induced changes, correlation of immunotherapy efficacy and obesity is unpredictable. The overall goal of this proposal is to understand how the systemic and tumor immune microenvironment differences in obesity affect immunotherapy efficacy and to determine the role of tumor associated macrophages in immunotherapy efficacy. We have established a diet- induced obesity mouse model using a 60% kcal high-fat diet in C57BL/6 mice that is compatible with the MC38 colon cancer cell line, which is well-published as an immunoresponsive cell line to immune checkpoint blockade treatments. In Specific Aim 1, we will investigate how systemic and tumor immune microenvironment differences in obese and lean mice affect anti-PD-1 treatment efficacy and the systemic and tumor immune microenvironment post-treatment. In Specific Aim 2, we will determine the role of tumor associated macrophages in anti-PD-1 treatment efficacy by treating mice with a mannosylated delivery system for I?Ba siRNA, which activates the NF?B pathway and creates pro-inflammatory, M1 macrophages. For both of these aims we will characterize the tumor immune microenvironments using flow cytometry and immunohistochemistry, while systemic cytokines and adipokines will be analyzed using ELISA and Luminex assays. Together, these studies will define the role of obesity and tumor associated macrophages in immune checkpoint blockade efficacy and will suggest if translational mouse models should include obese murine studies before new immunotherapy drugs are used clinically. Further, these results will identify methods in which to improve immunotherapy efficacy. Finally, these results will reveal if obesity is an important characteristic to consider when treating cancer patients with immunotherapy.